Our long term goal of this proposal will be to elucidate the insulin function at the molecular level. This grant application will cover two major projects: (1) to purify human receptor subunits so as to determine the partial amino acid sequence, (2) to clone the cDNA coded for the receptor so as to determine the total primary sequence of the receptor. Insulin receptor has been studied mainly by using rat liver and adipocyte membranes. Although the purified receptor has not proven to be homogeneous and has not been obtained enough to characterize, there is plenty of evidence that the 135,000 dalton component is a subunit of the receptor. However, human insulin receptor seems to have different subunits. The discrepancy would be solved if we purify the receptor and analyze it by the affinity-labeling methods. After establishing a large scale purification by applying our recently developed insulin-dextran affinity adsorbent, we will determine the partial amino acid sequence of the receptor, and clone cDNA for the receptor to deduce the total amino acid sequence by the DNA sequence and to produce a large amount of subunits in E. coli. After this accomplishment, it will be possible to pursue our future projects such as studies on the structure of the receptor, the interaction between the receptor and insulin, and gene organization and regulation of the receptor. We will prepare monoclonal antibodies against receptor subunits by using hybridoma technology, which would be a great help for the purification and future projects. These experiments will give us a deep understanding in the pathogenesis of diabetes mellitus and may open up a new way to treat the disease.